A number of environmental toxicants were shown in the previous grant period to promote the epigenetic transgenerational inheritance of adult onset disease including the fungicide vinclozolin, pesticides, plastics (BPA and phthalates), dioxin, hydrocarbons, and DDT. The ability of environmental compounds (e.g. endocrine disruptors) to promote transgenerational disease phenotypes is anticipated to be a critical aspect of adult onset disease etiology. The transgenerational inheritance phenotype requires the heritable epigenetic alterations of the germline. The current proposal is designed to further investigate the molecular and developmental aspects of the epigenetic transgenerational inheritance phenomenon and assess the potential use of epigenetic biomarkers for exposure and disease. The general hypothesis tested is that transient fetal exposure around the period of gonadal sex determination to environmental toxicants (i.e. vinclozolin) promotes the reprogramming of the epigenome (i.e. DNA methylation) of the male germline that then transmits an imprinted-like epigenome to subsequent generations to induce the epigenetic transgenerational inheritance of adult onset disease (e.g. male infertility). The previous grant period focused on the analysis of the epigenetic alteration in the F3 generation sperm using promoter associated differential DNA methylation regions (epimutations), as well as documented the epigenetic and transcriptome changes in the F3 generation vinclozolin lineage primordial germ cells. The molecular etiology of somatic cell changes associated with ovary and testis disease was established. The current proposal further investigates the molecular and developmental aspects of the germline mediated epigenetic transgenerational inheritance of disease. The experimental approach to test the above hypothesis consists of the following specific aims: Aim 1) Determine the developmental and generational aspects of the genome-wide germline epimutations. Aim 2) Determine the genomic features associated with the epimutations and potential genetic alterations involved in the epigenetic transgenerational inheritance phenomenon. Aim 3) Determine the optimal exposure parameters of the environmental induced transgenerational model and correlate epigenetic biomarkers with exposure and disease. Completion of the proposed research will determine the cascade of epigenetic events involved in germ cell development that generates the sperm epimutations that transmit the epigenetic transgenerational inheritance of disease phenotypes. The protection of the sperm epimutations from DNA methylation erasure is anticipated in early embryonic development. The proposed research will further elucidate the molecular and developmental aspects of environmentally induced epigenetic transgenerational inheritance of disease and the potential use of epigenetic biomarkers for ancestral exposure and disease.